ROLE OF CAMP-DEPENDENT PROTEIN KINASE IN GROWTH CONTROL

我们的长期目标是通过深入研究cAMP依赖性蛋白激酶（PKA）的调节（R）和催化（C）亚基的结构、功能和表达来了解PKA对细胞生长的调节。 PKA的两种亚型PKA-I和PKA-II共享一个共同的催化（C）亚基，但分别含有不同的调节（R）亚基RI和RII。 已知PKA-I及其调节亚基RIa的表达在人癌细胞系、原发性肿瘤和转化后的细胞中增加。 （1）反义策略的应用：利用RIa反义寡核苷酸或反义RIa基因的过表达来证明序列特异性抑制RIa基因表达和体内肿瘤生长抑制。反义RNA导致RIa的缺失导致RIIb的快速增加 PKA-IIb 全酶 脉冲追踪实验表明，RIIb 在反义处理的细胞中，蛋白质的半衰期增加了3-6倍。 因此，RIIb 在全酶复合物中的稳定化，表现出增加的半衰期。 通过这种生物化学适应，在反义处理的癌细胞中，PKA-I与PKA-II的比例变化到与正常细胞相似。 (2)重组基因技术：逆转录病毒载体介导的野生型和点突变RIa、RIIa、RIIb的过表达 （3）细胞外PKA（ECPKA）的发现：PKA的游离催化（C）亚基在多种癌细胞的条件培养液和癌症患者血清中分泌，ECPKA表达较正常血清上调10倍。 重要的是，本研究提供了通过改变细胞内PKA-I和PKA-II的表达来调节ECPKA表达的方法。 即，（1）RIa的过表达 （2）RIIb的过表达下调了细胞内PKA-I的表达，逆转了转化表型，下调了ECPKA的表达;和（3）阻止肉豆蔻酰化的Ca基因突变允许细胞内PKA上调但阻断ECPKA增加，表明Ca的N-末端肉豆蔻基是ECPKA表达所必需的。 进一步显示，与非依赖性乳腺癌相比，ECPKA上调在维持乳腺癌依赖性（正常细胞性质）的癌细胞中减少，例如乳腺癌依赖性乳腺癌。 这些观察结果表明ECPKA表达的这种现象可以为癌症诊断、预后和肿瘤依赖性检测（乳腺癌）提供创新方法。

Our long term goal is to understand the regulation of cell growth by cAMP-dependent protein kinase (PKA) by an in-depth investigation of the structure, function and expression of the regulatory (R) and catalytic (C) subunits of PKA. Two isoforms of PKA, PKA-I and PKA-II, share a common catalytic (C) subunit but contain distinct regulatory (R) subunits, RI and RII, respectively. It is known that expression of PKA-I and its regulatory subunit, RIa is increased in human cancer cell lines, in primary tumors, and in cells after transformation. Our studies revealed the following: (1) Use of antisense strategy: RIa antisense oligodeoxynucleotide or antisense RIa gene overexpression is used to demonstrate the sequence-specific inhibition of RIa gene expression and in vivo tumor growth inhibition. The loss of RIa by the antisense resulted in rapid increase in RIIb and PKA-IIb holoenzyme. Pulse-chase experiments demonstrated that RIIb protein increased its half-life 3-6 fold in antisense treated cells. Thus, RIIb in the holoenzyme complex is stabilized, exhibiting an increased half-life. Through this biochemical adaptation, in the antisense-treated cancer cells, the ratio of PKA-I to PKA-II changes to that similar to that of normal cells.; (2) Recombinant gene technology: Retroviral-vector mediated overexpression of wild type and point-mutated RIa, RIIa, RIIb and C subunits of PKA to demonstrate distinctive roles of the R subunit isoforms in cell growth, differentiation, and reverse transformation; and (3) Discovery of extracellular PKA (ECPKA):the free catalytic (C) subunit of PKA is excreted in conditioned medium of various cancer cells, and in serum of cancer patients; the ECPKA expression was upregulated 10-fold as compared with normal serum. Importantly, this study provided the means of modulating the ECPKA expression by changing expression of the intracellular PKA-I and PKA-II. Namely, (1) overexpression of RIa in expression vector, which upregulates intracellular PKA-I, can markedly upregulate ECPKA expression; (2) overexpression of RIIb, which downregulates PKA-I in the cell and reverts the transformed phenotype, downregulates ECPKA; and (3) A mutation in the Ca gene that prevents myristylation allows the intracellular PKA upregulation but blocks the ECPKA increase, suggesting that the N-terminal myristyl group of Ca is required for ECPKA expression. It was further shown that ECPKA upregulation is reduced in cancer cells maintaining hormone-dependency (a normal cell property), such as hormone-dependent breast cancer as compared to the hormone-independent breast cancer. These observations indicate that this phenomenon of ECPKA expression can provide innovative approach to cancer diagnosis, prognosis, and hormone-dependence detection (breast cancer).